Fabrication of chitin-fibrin hydrogels to construct the 3D artificial extracellular matrix scaffold for vascular regeneration and cardiac tissue engineering.

As the cornerstone of tissue engineering and regeneration medicine research, developing a cost-effective and bionic extracellular matrix (ECM) that can precisely modulate cellular behavior and form functional tissue remains challenging. An artificial ECM combining polysaccharides and fibrillar proteins to mimic the structure and composition of natural ECM provides a promising solution for cardiac tissue regeneration. In this study, we developed a bionic hydrogel scaffold by combining a quaternized ß-chitin derivative (QC) and fibrin-matrigel (FM) in different ratios to mimic a natural ECM. We evaluated the stiffness of those composite hydrogels with different mixing ratios and their effects on the growth of human umbilical vein endothelial cells (HUVECs). The optimal hydrogels, QCFM1 hydrogels were further applied to load HUVECs into nude mice for in vivo angiogenesis. Besides, we encapsulated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) into QCFM hydrogels and employed 3D bioprinting to achieve batch fabrication of human-engineered heart tissue (hEHT). Finally, the myocardial structure and electrophysiological function of hEHT were evaluated by immunofluorescence and optical mapping. Designed artificial ECM has a tunable modulus (220-1380 Pa), which determines the different cellular behavior of HUVECs when encapsulated in these. QCFM1 composite hydrogels with optimal stiffness (800 Pa) and porous architecture were finally identified, which could adapt for in vitro cell spreading and in vivo angiogenesis of HUVECs. Moreover, QCFM1 hydrogels were applied in 3D bioprinting successfully to achieve batch fabrication of both ring-shaped and patch-shaped hEHT. These QCFM1 hydrogels-based hEHTs possess organized sarcomeres and advanced function characteristics comparable to reported hEHTs. The chitin-derived hydrogels are first used for cardiac tissue engineering and achieve the batch fabrication of functionalized artificial myocardium. Specifically, these novel QCFM1 hydrogels provided a reliable and economical choice serving as ideal ECM for application in tissue engineering and regeneration medicine.

Biofabrication and Monitoring of a 3D Printed Skin Model for Melanoma.

There is an unmet need for in vitro cancer models that emulate the complexity of human tissues. 3D-printed solid tumor micromodels based on decellularized extracellular matrices (dECMs) recreate the biomolecule-rich matrix of native tissue. Herein a 3D in vitro metastatic melanoma model that is amenable for drug screening purposes and recapitulates features of both the tumor and the skin microenvironment is described. Epidermal, basement membrane, and dermal biocompatible inks are prepared by means of combined chemical, mechanical, and enzymatic processes. Bioink printability is confirmed by rheological assessment and bioprinting, and bioinks are subsequently combined with melanoma cells and dermal fibroblasts to build complex 3D melanoma models. Cells are tracked by confocal microscopy and surface-enhanced Raman spectroscopy (SERS) mapping. Printed dECMs and cell tracking allow modeling of the initial steps of metastatic disease, and may be used to better understand melanoma cell behavior and response to drugs.

A Comprehensive Literature Review on Advancements and Challenges in 3D Bioprinting of Human Organs: Ear, Skin, and Bone.

The field of 3D bioprinting is rapidly emerging within the realm of regenerative medicine, offering significant potential in dealing with the issue of organ shortages. Despite being in its early stages, it has the potential to replicate tissue structures accurately, providing new potential solutions for reconstructive surgery. This review explores the diverse applications of 3D bioprinting in regenerative medicine, pharmaceuticals, and the food industry, specifically focusing on ear, skin, and bone tissues due to their unique challenges and implications in the field. Significant progress has been made in cartilage and bone scaffold fabrication in ear reconstruction, yet challenges in functional maturation persist. Recent advancements highlight the potential for patient-specific ear substitutes, emphasizing the need for extensive clinical trials. In skin regeneration, 3D bioprinting addresses limitations in existing models, offering opportunities for improved wound healing and realistic skin models. While challenges exist, progress in biomaterials and in-situ bioprinting holds promise. In bone regeneration, 3D bioprinting presents personalized solutions for defects, but scaffold design refinement and addressing regulatory and ethical considerations are crucial. The transformative potential of 3D bioprinting in the field of medicine holds the promise of redefining therapeutic approaches and delivering personalized treatments and functional tissues. Interdisciplinary collaboration is essential for fully realizing the capabilities of 3D bioprinting. This review provides a detailed analysis of current methodologies, challenges, and prospects in 3D bioprinting for ear, skin, and bone tissue regeneration.

Advanced biomedical and electronic dual-function skin patch created through microfluidic-regulated 3D bioprinting.

Artificial skin involves multidisciplinary efforts, including materials science, biology, medicine, and tissue engineering. Recent studies have aimed at creating skins that are multifunctional, intelligent, and capable of regenerating tissue. In this work, we present a specialized 3D printing ink composed of polyurethane and bioactive glass (PU-BG) and prepare dual-function skin patch by microfluidic-regulated 3D bioprinting (MRBP) technique. The MRBP endows the skin patch with a highly controlled microstructure and superior strength. Besides, an asymmetric tri-layer is further constructed, which promotes cell attachment and growth through a dual transport mechanism based on hydrogen bonds and gradient structure from hydrophilic to superhydrophilic. More importantly, by combining the features of biomedical skin with electronic skin (e-skin), we achieved a biomedical and electronic dual-function skin patch. In vivo experiments have shown that this skin patch can enhance hemostasis, resist bacterial growth, stimulate the regeneration of blood vessels, and accelerate the healing process. Meanwhile, it also mimics the sensory functions of natural skin to realize signal detection, where the sensitivity reached up to 5.87 kPa-1, as well as cyclic stability (over 500 cycles), a wide detection range of 0-150 kPa, high pressure resolution of 0.1 % under the pressure of 100 kPa. This work offers a versatile and effective method for creating dual-function skin patches and provide new insights into wound healing and tissue repair, which have significant implications for clinical applications.

Single-Step 3D Bioprinting of Alginate-Collagen Type I Hydrogel Fiber Rings to Promote Angiogenic Network Formation.

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients. The spatial arrangement of the SV-ARBECs within the rings promotes spontaneous angiogenic network formation and the constrained deposition of cells within the hydrogel matrix facilitates tissue-like organized vascular-like network development. This approach provides a platform that can be adapted to many different endothelial cell types and leveraged to better understand the mechanisms driving angiogenesis and vascular-network formation in 3D bioprinted constructs supporting the development of more complex tissue and disease models for advancing drug discovery, tissue engineering, and regenerative medicine applications.

Application of 3D bioprinting technology apply to assessing Dangguiniantongtang (DGNT) decoctions in arthritis.

The aim of this study was to develop a three-dimensional (3D) cell model in order to evaluate the effectiveness of a traditional Chinese medicine decoction in the treatment of arthritis. Chondrocytes (ATDC5) and osteoblasts (MC3T3-E1) were 3D printed separately using methacryloyl gelatin (GelMA) hydrogel bioinks to mimic the natural 3D cell environment. Both cell types showed good biocompatibility in GelMA. Lipopolysaccharide (LPS) was added to the cell models to create inflammation models, which resulted in increased expression of inflammatory factors IL-1ß, TNF-α, iNOS, and IL-6, and decreased expression of cell functional genes such as Collagen II (COLII), transcription factor SOX-9 (Sox9), Aggrecan, alkaline phosphatase (ALP), RUNX family transcription factor 2 (Runx2), Collagen I (COLI), Osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2). The created inflammation model was then used to evaluate the effectiveness of Dangguiniantongtang (DGNT) decoctions. The results showed that DGNT reduced the expression of inflammatory factors and increased the expression of functional genes in the cell model. In summary, this study established a 3D cell model to assess the effectiveness of traditional Chinese medicine (TCM) decoctions, characterized the gene expression profile of the inflammatory state model, and provided a practical reference for future research on TCM efficacy evaluation for arthritis treatment.

Scaffolding fundamentals and recent advances in sustainable scaffolding techniques for cultured meat development.

In cultured meat (CM) production, Scaffolding plays an important role by aiding cell adhesion, growth, differentiation, and alignment. The existence of fibrous microstructure in connective and muscle tissues has attracted considerable interest in the realm of tissue engineering and triggered the interest of researchers to implement scaffolding techniques. A wide array of research efforts is ongoing in scaffolding technologies for achieving the real meat structure on the principality of biomedical research and to replace serum free CM production. Scaffolds made of animal-derived biomaterials are found efficient in replicating the extracellular matrix (ECM), thus focus should be paid to utilize animal byproducts for this purpose. Proper identification and utilization of plant-derived scaffolding biomaterial could be helpful to add diversified options in addition to animal derived sources and reduce in cost of CM production through scaffolds. Furthermore, techniques like electrospinning, modified electrospinning and 3D bioprinting should be focused on to create 3D porous scaffolds to mimic the ECM of the muscle tissue and form real meat-like structures. This review discusses recent advances in cutting edge scaffolding techniques and edible biomaterials related to structured CM production.

3D bioprinted mesenchymal stem cell laden scaffold enhances subcutaneous vascularization for delivery of cell therapy.

Subcutaneous delivery of cell therapy is an appealing minimally-invasive strategy for the treatment of various diseases. However, the subdermal site is poorly vascularized making it inadequate for supporting engraftment, viability, and function of exogenous cells. In this study, we developed a 3D bioprinted scaffold composed of alginate/gelatin (Alg/Gel) embedded with mesenchymal stem cells (MSCs) to enhance vascularization and tissue ingrowth in a subcutaneous microenvironment. We identified bio-ink crosslinking conditions that optimally recapitulated the mechanical properties of subcutaneous tissue. We achieved controlled degradation of the Alg/Gel scaffold synchronous with host tissue ingrowth and remodeling. Further, in a rat model, the Alg/Gel scaffold was superior to MSC-embedded Pluronic hydrogel in supporting tissue development and vascularization of a subcutaneous site. While the scaffold alone promoted vascular tissue formation, the inclusion of MSCs in the bio-ink further enhanced angiogenesis. Our findings highlight the use of simple cell-laden degradable bioprinted structures to generate a supportive microenvironment for cell delivery.

Three-Dimensional Printing in Breast Reconstruction: Current and Promising Applications.

Three-dimensional (3D) printing is dramatically improving breast reconstruction by offering customized and precise interventions at various stages of the surgical process. In preoperative planning, 3D imaging techniques, such as computer-aided design, allow the creation of detailed breast models for surgical simulation, optimizing surgical outcomes and reducing complications. During surgery, 3D printing makes it possible to customize implants and precisely shape autologous tissue flaps with customized molds and scaffolds. This not only improves the aesthetic appearance, but also conforms to the patient's natural anatomy. In addition, 3D printed scaffolds facilitate tissue engineering, potentially favoring the development and integration of autologous adipose tissue, thus avoiding implant-related complications. Postoperatively, 3D imaging allows an accurate assessment of breast volume and symmetry, which is crucial in assessing the success of reconstruction. The technology is also a key educational tool, enhancing surgeon training through realistic anatomical models and surgical simulations. As the field evolves, the integration of 3D printing with emerging technologies such as biodegradable materials and advanced imaging promises to further refine breast reconstruction techniques and outcomes. This study aims to explore the various applications of 3D printing in breast reconstruction, addressing current challenges and future opportunities.

Propelling Minimally Invasive Tissue Regeneration With Next-Era Injectable Pre-Formed Scaffolds.

The growing aging population, with its associated chronic diseases, underscores the urgency for effective tissue regeneration strategies. Biomaterials play a pivotal role in the realm of tissue reconstruction and regeneration, with a distinct shift toward minimally invasive (MI) treatments. This transition, fueled by engineered biomaterials, steers away from invasive surgical procedures to embrace approaches offering reduced trauma, accelerated recovery, and cost-effectiveness. In the realm of MI tissue repair and cargo delivery, various techniques are explored. While in situ polymerization is prominent, it is not without its challenges. This narrative review explores diverse biomaterials, fabrication methods, and biofunctionalization for injectable pre-formed scaffolds, focusing on their unique advantages. The injectable pre-formed scaffolds, exhibiting compressibility, controlled injection, and maintained mechanical integrity, emerge as promising alternative solutions to in situ polymerization challenges. The conclusion of this review emphasizes the importance of interdisciplinary design facilitated by synergizing fields of materials science, advanced 3D biomanufacturing, mechanobiological studies, and innovative approaches for effective MI tissue regeneration.

Microgels for Cell Delivery in Tissue Engineering and Regenerative Medicine.

Microgels prepared from natural or synthetic hydrogel materials have aroused extensive attention as multifunctional cells or drug carriers, that are promising for tissue engineering and regenerative medicine. Microgels can also be aggregated into microporous scaffolds, promoting cell infiltration and proliferation for tissue repair. This review gives an overview of recent developments in the fabrication techniques and applications of microgels. A series of conventional and novel strategies including emulsification, microfluidic, lithography, electrospray, centrifugation, gas-shearing, three-dimensional bioprinting, etc. are discussed in depth. The characteristics and applications of microgels and microgel-based scaffolds for cell culture and delivery are elaborated with an emphasis on the advantages of these carriers in cell therapy. Additionally, we expound on the ongoing and foreseeable applications and current limitations of microgels and their aggregate in the field of biomedical engineering. Through stimulating innovative ideas, the present review paves new avenues for expanding the application of microgels in cell delivery techniques.

Blood vessels in a dish: the evolution, challenges, and potential of vascularized tissues and organoids.

Vascular pathologies are prevalent in a broad spectrum of diseases, necessitating a deeper understanding of vascular biology, particularly in overcoming the oxygen and nutrient diffusion limit in tissue constructs. The evolution of vascularized tissues signifies a convergence of multiple scientific disciplines, encompassing the differentiation of human pluripotent stem cells (hPSCs) into vascular cells, the development of advanced three-dimensional (3D) bioprinting techniques, and the refinement of bioinks. These technologies are instrumental in creating intricate vascular networks essential for tissue viability, especially in thick, complex constructs. This review provides broad perspectives on the past, current state, and advancements in key areas, including the differentiation of hPSCs into specific vascular lineages, the potential and challenges of 3D bioprinting methods, and the role of innovative bioinks mimicking the native extracellular matrix. We also explore the integration of biophysical cues in vascularized tissues in vitro, highlighting their importance in stimulating vessel maturation and functionality. In this review, we aim to synthesize these diverse yet interconnected domains, offering a broad, multidisciplinary perspective on tissue vascularization. Advancements in this field will help address the global organ shortage and transform patient care.

3D Bioprinting of Artificial Skin Substitute with Improved Mechanical Property and Regulated Cell Behavior through Integrating Patterned Nanofibrous Films.

Three-dimensional (3D) bioprinting has advantages for constructing artificial skin tissues in replicating the structures and functions of native skin. Although many studies have presented improved effect of printing skin substitutes in wound healing, using hydrogel inks to fabricate 3D bioprinting architectures with complicated structures, mimicking mechanical properties, and appropriate cellular environments is still challenging. Inspired by collagen nanofibers withstanding stress and regulating cell behavior, a patterned nanofibrous film was introduced to the printed hydrogel scaffold to fabricate a composite artificial skin substitute (CASS). The artificial dermis was printed using gelatin-hyaluronan hybrid hydrogels containing human dermal fibroblasts with gradient porosity and integrated with patterned nanofibrous films simultaneously, while the artificial epidermis was formed by seeding human keratinocytes upon the dermis. The collagen-mimicking nanofibrous film effectively improved the tensile strength and fracture resistance of the CASS, making it sewable for firm implantation into skin defects. Meanwhile, the patterned nanofibrous film also provided the biological cues to guide cell behavior. Consequently, CASS could effectively accelerate the regeneration of large-area skin defects in mouse and pig models by promoting re-epithelialization and collagen deposition. This research developed an effective strategy to prepare composite bioprinting architectures for enhancing mechanical property and regulating cell behavior, and CASS could be a promising skin substitute for treating large-area skin defects.

FRESH-based 3D bioprinting of complex biological geometries using chitosan bioink.

Traditional three-dimensional (3D) bioprinting has always been associated with the challenge of print fidelity of complex geometries due to the gel-like nature of the bioinks. Embedded 3D bioprinting has emerged as a potential solution to print complex geometries using proteins and polysaccharides-based bioinks. This study demonstrated the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) 3D bioprinting method of chitosan bioink to 3D bioprint complex geometries. 4.5% chitosan was dissolved in an alkali solvent to prepare the bioink. Rheological evaluation of the bioink described its shear-thinning nature. The power law equation was fitted to the shear rate-viscosity plot. The flow index value was less than 1, categorizing the material as pseudo-plastic. The chitosan bioink was extruded into another medium, a thermo-responsive 4.5% gelatin hydrogel. This hydrogel provides support to the growing print structures while printing. After this, the 3D bioprinted structure was crosslinked with 60° C water to stabilize the structure. Using this method, we have 3D bioprinted complex biological structures like the human tri-leaflet heart valve, a section of a human right coronary arterial tree, a scale-down outer structure of the human kidney, and a human ear. Additionally, we have shown the mechanical tunability and suturability of the 3D bioprinted structures. This demonstrates the capability of the chitosan bioink and FRESH method for 3D bioprinting of biological models for surgical training and planning.

Evaluation of allylated gelatin as a bioink supporting spontaneous spheroid formation of HepG2 cells.

The spheroid culture system has gained significant attention as an effective in vitro model to mimic the in vivo microenvironment. Even though numerous studies were focused on developing spheroids, the structural organization of encapsulated cells within hydrogels remains a challenge. Allylated gelatin or GelAGE is used as a bioink due to its excellent physicochemical properties. In this study, GelAGE was evaluated for its capacity to induce spontaneous spheroid formation in encapsulated HepG2 cells. GelAGE was synthesized and characterized using 1HNMR spectroscopy and ninhydrin assay. Then the physicochemical and biological attributes of GelAGE hydrogel was examined. The results demonstrate that GelAGE has remarkable ability to induce the encapsulated cells to self-organize into spheroids.

(3D) Bioprinting-Next Dimension of the Pharmaceutical Sector.

To create a review of the published scientific literature on the benefits and potential perspectives of the use of 3D bio-nitrification in the field of pharmaceutics. This work was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for reporting meta-analyses and systematic reviews. The scientific databases PubMed, Scopus, Google Scholar, and ScienceDirect were used to search and extract data using the following keywords: 3D bioprinting, drug research and development, personalized medicine, pharmaceutical companies, clinical trials, drug testing. The data points to several aspects of the application of bioprinting in pharmaceutics were reviewed. The main applications of bioprinting are in the development of new drug molecules as well as in the preparation of personalized drugs, but the greatest benefits are in terms of drug screening and testing. Growth in the field of 3D printing has facilitated pharmaceutical applications, enabling the development of personalized drug screening and drug delivery systems for individual patients. Bioprinting presents the opportunity to print drugs on demand according to the individual needs of the patient, making the shape, structure, and dosage suitable for each of the patient's physical conditions, i.e., print specific drugs for controlled release rates; print porous tablets to reduce swallowing difficulties; make transdermal microneedle patches to reduce patient pain; and so on. On the other hand, bioprinting can precisely control the distribution of cells and biomaterials to build organoids, or an Organ-on-a-Chip, for the testing of drugs on printed organs mimicking specified disease characteristics instead of animal testing and clinical trials. The development of bioprinting has the potential to offer customized drug screening platforms and drug delivery systems meeting a range of individualized needs, as well as prospects at different stages of drug development and patient therapy. The role of bioprinting in preclinical and clinical testing of drugs is also of significant importance in terms of shortening the time to launch a medicinal product on the market.

Multifaceted cancer alleviation by cowpea mosaic virus in a bioprinted ovarian cancer peritoneal spheroid model.

Ovarian cancer (OvCa) is a leading cause of mortality among gynecological malignancies and usually manifests as intraperitoneal spheroids that generate metastases, ascites, and an immunosuppressive tumor microenvironment. In this study, we explore the immunomodulatory properties of cowpea mosaic virus (CPMV) as an adjuvant immunotherapeutic agent using an in vitro model of OvCa peritoneal spheroids. Previous findings highlighted the potent efficacy of intratumoral CPMV against OvCa in mouse tumor models. Leveraging the precision control over material deposition and cell patterning afforded by digital-light-processing (DLP) based bioprinting, we constructed OvCa-macrophage spheroids to mimic peritoneal spheroids using gelatin methacrylate (GelMA), a collagen-derived photopolymerizable biomaterial to mimic the extracellular matrix. Following CPMV treatment, bioprinted spheroids exhibited inhibited OvCa progression mediated by macrophage activation. Our analysis indicates that CPMV regulates and activates macrophage to both induce OvCa cell killing and restore normal cell-cell junctions. This study deepened our understanding of the mechanism of CPMV intratumoral immunotherapy in the setting of OvCa. This study also highlights the potential of studying immunotherapies using high throughput tissue models via DLP bioprinting.

Lutein Modulates Cellular Functionalities and Regulates NLRP3 Inflammasome in a H2O2-Challenged Three-Dimensional Retinal Pigment Epithelium Model.

Excessive hydrogen peroxide (H2O2) generated during retinal cell metabolic activity could lead to oxidative degeneration of retinal pigment epithelium (RPE) tissue, a specific pathological process implicated in various retinal diseases resulting in blindness, which can be mitigated by taking dietary antioxidants to prevent inflammation and impaired cellular dysfunction. This study tested the hypothesis that damages induced by oxidative stresses can be mitigated by lutein in a H2O2-challenged model, which was based on an ARPE-19 cell monolayer cultured on three-dimensional (3D)-printed fibrous scaffolds. Pretreating these models with lutein (0.5 µM) for 24 h can significantly lower the oxidative stress and maintain phagocytosis and barrier function. Moreover, lutein can modulate the NLRP3 inflammasome, leading to a ∼40% decrease in the pro-inflammatory cytokine (IL-1ß and IL-18) levels. Collectively, this study suggests that the 3D RPE model is an effective tool to examine the capability of lutein to modulate cellular functionalities and regulate NLRP3 inflammation.

3D printing and bioprinting in the battle against diabetes and its chronic complications.

Diabetes is a metabolic disorder characterized by high blood sugar. Uncontrolled blood glucose affects the circulatory system in an organism by intervening blood circulation. The high blood glucose can lead to macrovascular (large blood vessels) and microvascular (small blood vessels) complications. Due to this, the vital organs (notably brain, eyes, feet, heart, kidneys, lungs and nerves) get worsen in diabetic patients if not treated at the earliest. Therefore, acquiring treatment at an appropriate time is very important for managing diabetes and other complications that are caused due to diabetes. The root cause for the occurrence of various health complications in diabetic patients is the uncontrolled blood glucose levels. This review presents a consolidated account of the applications of various types of three-dimensional (3D) printing and bioprinting technologies in treating diabetes as well as the complications caused due to impaired blood glucose levels. Herein, the development of biosensors (for the diagnosis), oral drug formulations, transdermal drug carriers, orthotic insoles and scaffolds (for the treatment) are discussed. Next to this, the fabrication of 3D bioprinted organs and cell-seeded hydrogels (pancreas engineering for producing insulin and bone engineering for managing bone defects) are explained. As the final application, 3D bioprinting of diabetic disease models for high-throughput screening of ant-diabetic drugs are discussed. Lastly, the challenges and future perspective associated with the use of 3D printing and bioprinting technologies against diabetes and its related chronic complications have been put forward.

Digitalization in Cranial Reconstruction: Revolutionizing Precision and Innovation.

Cranioplasty for cranial defects can be complex and challenging in composite defects. The intricate 3D structure of the craniofacial skeleton poses various difficulties encountered in surgical reconstruction. The continuous progress in computer-aided design and computer-aided manufacturing design, and fabrication technology has led to a growing array of applications for visual analog scale and 3D printing in craniofacial surgery, encompassing preoperative assessment, the creation of cutting guides, and the development of custom implants and stereolithographic models. Within this review, the authors detail the present and developing applications of virtual surgical planning, 3D bioprinting, augmented reality, and virtual reality in craniofacial reconstruction.